Abstract

Circulating tumor cells (CTCs) shed from the primary tumor mass and circulating in the bloodstream of patients are believed to be vital to understand of cancer metastasis and progression. Capture and release of CTCs for further enumeration and molecular characterization holds the key for early cancer diagnosis, prognosis and therapy evaluation. However, detection of CTCs is challenging due to their rarity, heterogeneity and the increasing demand of viable CTCs for downstream biological analysis. Nanotopographic biomaterial-based microfluidic systems are emerging as promising tools for CTC capture with improved capture efficiency, purity, throughput and retrieval of viable CTCs. This review offers a brief overview of the recent advances in this field, including CTC detection technologies based on nanotopographic biomaterials and relevant nanofabrication methods. Additionally, the possible intracellular mechanisms of the intrinsic nanotopography sensitive responses that lead to the enhanced CTC capture are explored.

FIGURES IN THIS ARTICLE

Introduction

Cancer related disease treatment remains a challenge worldwide since cancer was first identified in the 19th century [1]. Increasing evidence has revealed that cancer metastasis plays a critical role in the process of disease relapse and exacerbation [2-4]. During metastasis, an extremely small group of tumor cells, termed CTCs, can shed from the primary tumor mass and enter the bloodstream. They then circulate through the peripheral blood, settling at other sites and tissues in the human body and developing further metastases [5-8]. Clinical investigations of CTCs have provided a mountain of indications about their role in cancer metastasis and suggested that the level of CTCs in patients indicated different survival rates [9-16]. Recent molecular characterization of CTCs has also provided evidences concerning the tumor-derived nature of CTCs [17]. Increasing our understanding of this rare cell population will serve to probe the mechanism of cancer development and potentially offer new cancer treatments. In addition, accessing the peripheral blood from patients to collect CTCs for disease progression and treatment efficacy evaluation is noninvasive and effective [18,19]. Therefore, collection and analysis of CTCs is key to gain new insight into the biology of cancer metastasis while acting as a “liquid biopsy” that noninvasively implements cancer prognosis, detection and treatment.

Although the analysis of CTCs hold great promise in uncovering the metastatic mechanism and identifying therapeutic targets, it is still highly challenging to extract CTCs from patients with considerable efficiency and purity [20]. There is approximately only one CTC per ×109 blood cells in a cancer patient's bloodstream, indicating both the extreme rarity of CTCs and the highly efficient and sensitive platforms required for their detection. Ideally, the new CTC capture techniques would achieve a high recovery rate and specificity for accurate enumeration that would reflect cancer progression. They should also be able to release the captured CTCs for downstream biological analysis. Furthermore, CTCs have been observed to be heterogeneous, there are no unique and effective biologic markers for identification of CTCs for in all cancer patients [21-23]. Epithelial cell adhesion molecule (EpCAM), for example, have been long applied as surface marker for the capture of CTCs. However, some populations of CTCs have a low expression of EpCAM on the cell surface, suggesting that use of the EpCAM antibody alone might not be sufficient to identify and capture these CTC subpopulations [24,25]. Technologies making use of EpCAM for CTC capture thus risk omitting important information. As such, CTC isolation technologies effective for most types of cancer patients and cancers at different stages are highly demanded. To address the molecular characterization of CTCs, captured CTCs are expected to be viable upon release, or may be cultured and expanded directly inside the CTC capture device. Yet, due to the vulnerability of CTCs, releasing viable CTCs without harming their cellular intactness is a technical challenging [26]. The short life of CTCs poses additional difficulties in conducting high throughput separation.

To date, a large array of methods, especially microfluidics technology, has been developed to isolate CTCs from patients' blood, including those making use of the physical and biological differences between CTCs and normal blood cells [27-30]. For example, microfilter structures and hydrodynamic separation schemes have been widely used to distinguish CTCs from normal blood cells based on their size difference from normal blood cells [31-42]. Other physical features, such as surface charge have been exploited as well [43-49]. These physical feature-based CTC isolation methods conserve CTC morphological and biological intactness, but purity may be compromised due to the heterogeneous nature of CTCs and the extremely large number of background cells. Immunoaffinity methods, like the Cellsearch system approved by the Food and Drug Administration for clinical use, take advantages of functional antigens, e.g., EpCAM and prostate-specific membrane antigen expressed on CTC surfaces. This helps to improve the CTC capture specificity [50-53]. However, the release processes of these methods can destroy CTC intactness, an adverse outcome for CTC profiling. Besides, CTCs may experience epithelial-to-mesenchymal transition (EMT) and thereby lose epithelial features [54-56]. Aptamers are emerging as an alternative to antibodies for CTC capture and they are compatible with many different CTC isolation platforms [57-66]. Although having their individual virtues, new approaches must be developed which address the problems that these aforementioned methods have circumvented.

Nowadays, the increasing development in nanotechnology and materials sciences has provided great prospects for CTC capture evolution [67]. Particularly, nano-engineered biomaterials with nanoscale topographic cues have been demonstrated as promising solutions for CTC isolation and detection. in vivo, cancer cells reside in a distinct micro-environment, or called the “cancer cell niche,” where a diverse array of environmental factors such as mechanical signals and, adhesive and soluble factor gradients contributes to the overall control of CTC phenotypes and activities [68]. During metastatic progression, cancer cells also encounter complex biophysical environments consisting of different degrees of extracellular matrix (ECM) cross-linking, topology, and mechanical heterogeneity [68]. They are also exposed to shear flow and interstitial pressure. In response to the micro-environment, cancer cells display adaptive phenotypic properties so to facilitate functional behaviors including proliferation, EMT, and invasive and metastatic activities. Among these biophysical factors, ECM nanotopography has been proven to be a critical cue in regulating cancer cell behaviors, and nanotopography-based methods have recently been developed for the capture of CTCs. Various nanotopographic structures in the in vivo ECM such as fibers consisting of filamentous proteins with dimensions ranging from a few to hundreds of nanometers, have important effects on the functions of cancer cells [69-73]. Additionally, nanoscale adhesive molecules such as integrins at the cell membrane have been demonstrated through cellular probing of extracellular nanotopographic features [74]. Because of the similarity between an in vivo ECM and a nanotopographic substrate, artificial nanostructures which mimic the natural nanostructure of the ECM can be fabricated that promote the affinity between cells and substrates [75]. CTC capture purity can be further improved by combining nanostructures with antibodies and/or aptamers. The larger surface to volume ratio of nanotopographic substrates provide more space to modify them with antibodies and aptamers to enhance capture efficiency [76]. Well-developed nanofabrication technologies also facilitate the availability of various nanostructures. Different nanostructures, such as nanopillars/wires, nanofibers, and random nanoroughness have been applied for CTC capture and they demonstrated competitive advantages [64,65,77-89] (Fig. 1 and Table 1). Nanotopography cues, in combination with previously discussed CTC capture methods, would provide favorable solutions for the capture and study of CTCs. The purpose of this review is to provide a summary of the emerging technologies utilizing nanotopographic cues along with possible mechanisms for the capture and release of CTCs. Furthermore, different nanostructure fabrication methods will be reviewed and potential nanostructures that can be further investigated will also be touched upon.

Nanotopography-Enhanced Capture of CTCs.

The ECM and other micro/nanostructures on cell surfaces have long been known to be relevant to cellular fates by influencing cell adhesion migration, proliferation and differentiation [90-95]. Nanotopographic substrates, which mimic the characteristics of the nanotopographic cues in the local cell micro-environment, have been discovered to have strong interactions with nanoscale structures on the cell surface. This has led to broad researches about cell behavior and fate regulation through artificial nanostructures [96]. Particularly, recent studies have found that a great many types of cells, especially epithelial cells and cancer cells, have intrinsic sensitivity to the nanoscale surface topography and possess distinct adhesion properties to the nanostructured surfaces [90,97,98]. On the other hand, most blood cells including white blood cells (immune cells) and red blood cells are nonadhesive cells. These unique adhesion preferences of cancer cells for nanotopographic surfaces have been employed for the detection and enriching of CTCs from the background blood cells. Among these nanotopographic structures, nanopillars/wires, nanoroughened surfaces, and nanomaterials deposited on substrates such as nanofibers have been extensively investigated. Changing the nanoscale topography of substrates can largely increase the adhesion of the cancer cells to the substrate surface, and therefore increase the capture performance of CTCs.

Among the various nanostructures applied for CTC isolation and detection, nanopillars/wires coated with a capture agent are the most popular structures integrated into CTC capture devices. Nanopillar substrates achieved a higher efficiency compared to the capture performance of flat substrates [78]. The intense array of nanopillars/wires largely increases the ratio of surface area to volume and accordingly increases the area for more immobilization of capture antibody to enhance the interaction between CTCs and substrates. In addition, the enhanced affinity for nanotopographic substrate largely decreases the rolling velocity of CTCs and leads to greater capture efficiency. The first generation NanoVelcro chip system developed by Tseng's group is a typical CTC capture technologies utilizing nanopillar substrates [77]. In their first generation NanoVelcro chip system, a silicon-nanopillar (SiNP) array was integrated into microfluidics chips with fluidic chaotic mixer to further increase the interaction between CTCs and the SiNP array. Strikingly, this integrated capture system can process human whole blood and capture a diverse array of cancer cells including breast, prostate and carcinoma with high capture yield (>95%), viability and comparable throughput (up to 2 ml l−1) to the traditional methods. Silicon nanowires (SINWS), transparent quartz nanowire (QWN) arrays and micro/nanorod arrays were also prepared for the isolation of CTCs with good capture performances [79,99].

Recently, there is an increasing trend in using aptamer as a capture agent to replace EpCAM for the isolation of CTCs. Approaches using EpCAM biomarker may result in loss of some malignant CTCs because metastatic tumor cells may experience EMT and this could lead to the disappearance of epithelial features [100]. On the other hand, antibody-captured cancer cells were difficult to release for the downstream analysis. Hopefully, the limitations caused by antibody-based methods can be addressed by replacing anti-EpCAM antibody with aptamers. To this purpose, Tseng's group extended their first generation NanoVelcro chip systems by using aptamers as capture agents to modify SINWS [65]. Capture performance of nonsmall cell lung cancer cells spiked in healthy patient's blood using this chip systems and there was a >80% recovery rate. Meanwhile, the release process can be easily realized by enzymatic treatment and >85% release efficiency was achieved this way.

Apart from nanopillar/wire, various nanomaterials such as nanofibers have been employed for the isolation of CTCs [80-83]. Different from nanopillars/wires, which are extended in the vertical direction, nanofibers can be packed three-dimensionally (3D; both vertically, and horizontally) and form similar geometric orientations of the nanostructures in ECM [101]. In addition, nanofabrication technologies for nanofibrous matrices have been widely developed and applied in tissue engineering [102]. The dimension, alignment and desired pack density of nanofibers can be well controlled on substrates. Therefore, nanofibrous matrix would be an ideal candidate for application in nanotopography-based capture of CTCs. TiO2 nanofibers (TiNFs) with diameters of 100–300 nm embedded in silicon substrate were fabricated by electrospinning and used for detecting CTCs from colorectal and gastric cancer patients [80]. The nanofiber-based capture platform readily captured CTCs from the blood samples collected both from colorectal and gastric cancer patients with considerable capture efficiency (two out of three colorectal cancer patients and seven out of seven gastric cancer patients were detected with CTCs). Moreover, morphology differences of CTCs on flat silicon surface and TiNFs substrates were clearly observed. CTCs exhibited entirely outspread pseudopodia on TiNFs surface but showed a spherical conformation on the flat Si surface, confirming the enhanced interaction between cells and nanotopographic surfaces. Similarly, 3D structured biointerface consisting of micro/nanoscaled hierarchical fibrous network was applied to CTC captures [89]. Microfiber interfaces (MFs), nanofiber interfaces (NFs), and nanofibers/microbeads (NFs/MBs) were fabricated using polystyrene with random orientation on substrates and demonstrated 78.2%, 83.1%, and 89.2% recovery rates respectively in capturing human breast cancer cells, while only a 37.0% recovery rate was achieved using anti-EpCAM modified smooth substrates. Morphology differences were observed between 3D fibrous interfaces and smooth PS substrates but exhibited no significant differences among MFs, NFs, and NF/MB interfaces. The observation of extended long filopodia indicated that 3D fibrous interface may lead to the changing of cytoskeletal organization of CTCs.

Other nanomaterials, such as polymer nanodots, hallosite nanotubes have also been investigated [85,103]. Anti-EpCAM antibody modified graphene oxide nanosheets on a patterned gold surface were recently applied by Nagrath's group to build a planar nanostructured substrate for the capture of CTCs [87]. Micropillar structures (functionalized with capture antibodies) were commonly used in a microfluidic environment to increase the frequency that CTCs in the blood would encounter the functionalized surfaces and hence boost the capture performance. However, the micropillar structures normally have a height on the order of tens of microns and CTCs can be randomly captured at different vertical locations on the antibody-coated pillars. The variance in the vertical capture locations would cause difficulties in later imaging process. The varied focusing planes of the captured CTCs would prevent automatic scanning through a large surface area for the rapid detection of CTCs. However, the planar structure of nanosheets enabled ready imaging and culturing of captured CTCs, which is a major advantage over most 3D structures. The functionalized nanosheets embedded microfluidic chip was tested to be capable of achieving high recovery rate (up to 94%) as well as enhanced sensitivity (down to 2 cells per ml blood) for human whole blood. Importantly, due to the nanotopographic surfaces, this chip has proven to be an efficient tool for detecting CTCs from breast cancer patients at a very early stage.

Nanoroughened surface is another type of substrates that can be used for the enrichment of CTCs. Integrins, which are the cell adhesion molecules, can help cancer cells settle onto the nanostructured basement membrane. This way, an artificial nanoroughened surface that mimics the basement membrane can improve cell adhesion and growth [104,105]. Researches using surface nanotextured particle desorption mass spectrometry (PDMS) modified with aptamers have been conducted to capture human glioblastoma cells from a mixture of fibroblasts. It demonstrated one time higher capture efficiency than the glass slide and plain PDMS [64]. Although nanostructured surfaces modified with antibodies or aptamers can increase cell adhesion, investigations employing bare nanoroughened glass for CTC capture also achieved satisfactory results [106]. A label-free device with a nanoroughened glass substrate was prepared by Chen et al. for the isolation of CTCs independent of EpCAM expression on the cell surface [84]. This label-free platform captured both EpCAM positive and EpCAM negative breast cancer cells (e.g., MCF-7 and MDA-MB-231, respectively) with equivalently high performance (>90%). While for smooth glass substrates, only 13.9–22% capture yield were observed. Compared to the most of the other nanotopography-based methods which mostly have selectivity in cancer cell types due to the use of antibodies or aptamers, the label-free method using a bare glass could provide a better solution to capture cancer cells of different types and disease stages with a relatively low cost. Furthermore, captured cancer cells can be easily release from the nanoroughened substrates or cultured on the surfaces for long term studies. Similar to the observation before, adhesive strengths of cancer cells on nanoroughened glass substrates increased and further confirmed the enhanced adhesion of cells on nanoroughened glass substrates. Apart from the high capture yield, nanoroughened glass substrates can be readily assembled into microfluidic device and keep a high viability of captured cells by conducting in situ CTC culture and on-chip analysis. However, without antibody modification on substrates, capture specificity will be of concern because nonspecific attachment of blood cells was observed. Also, the adhesion of cancer cells to the nanoroughened glass might not as strong as the cancer cells binding to antibody. It may require a gentler processing of the sample than the immunoaffinity-based methods.

Release of CTCs From Nanotopographic Surfaces.

Molecular characterization of CTCs aims to get insights into the biology of CTCs and prompt the understanding of metastasis and cancer progression. Release of CTCs enables downstream molecular characterization and is gathering more and more attention. To conduct CTC analysis and get precise information from CTCs, the captured cells are expected to be maintained viable and released intact. As cells are vulnerable, the cell release process may risk destroying cell structures and keep us from probing biological information from them. For the enhanced capture of CTCs using nanotopographic substrates, this may pose additional challenges to the release of CTCs for following molecular analysis.

Release of CTCs from device using antibody for CTC capture often requires adding some biotins, like trypsin to destroy the linkage between cells and antibody [107]. Capture of CTCs using aptamers can not only achieve high recovery rate but also enable the release of viable cells [65]. Endonuclease is often used to degrade aptamers and research employed endonuclease to release CTCs obtain high rate of viable cells. Integrating nanotopography-enhanced capture of CTCs with other technologies, it is possible to retrieve CTCs without detach them from substrates. Tseng's group integrated the electrospun poly (lactic-co-glycolic acid) (PLGA) nanofibers to their NanoVelcro chip systems coupled with laser microdissection and developed the second generation NanoVelcro chip system [82,83]. Their system is capable of retrieving CTCs into single-CTC analysis by dissecting substrates where CTCs were identified. Their technology not only could achieve a recovery rate of 87% by capture human melanoma cells but also successfully carried out single circulating melanoma cells analysis and detected mutations matched that detected in patients' tumor biopsies. Using thermoresponsive materials is another trend for releasing CTCs from nanostructures [88,108]. Generally, thermoresponsive materials can be grafted into capture substrates and firstly used to capture CTCs. After the capture of CTCs, external stimulus like temperature change will accordingly change the structures of these grafted materials and lead to the release of CTCs. Poly (N-isopropylacrylamide) (PIPAAm) has been conjugated into the third NanoVelcro chip system by Tseng's group [88]. The conformation of PIPAAm is temperature-dependent. By adjusting temperature, backbones of PIPAAm will change between different states that enable or disable the contact of CTCs with capture agents. Minimum disruption to CTCs was observed when release them for molecular characterization. Combing the hydrophobic interactions with topographic interactions, Liu et al. were also able to reversibly capture and release cancer cells by taking advantages of thermoresponsive nanostructured surface [108]. Another type of materials that can be utilized is the electroactive materials, biotin-doped polypyrrole (Ppy) is an example of electroactive materials that has been employed for the capture and release of CTCs [109,110]. Application of electrical stimulus will release the modified biotin on Ppy and thus, release attached CTCs. Release of CTCs from nanotopographic substrates can also be realized by fabricating sacrificeable nanostructures where CTC attached. Biocompatible MnO2 nanosphere thin film and nanofibers, for example, were investigated for the capture and release of CTCs [81,111]. Besides achieving a capture yield of 80.9% spiked cancer cells, the release of CTCs can be easily achieved by dissolved MnO2 nanosphere thin film with oxalic acid at a very low concentration. Purity and viability of released CTCs can be up to 98% and 90%, respectively, indicated minimum destruction of low concentrated oxalic acid on captured CTCs [111]. Capture and release of CTCs using sacrificeable MnO2 nanofibers received similar results [81].

Potential Mechanisms for Nanotopography-Enhanced Capture of CTCs.

Although a lot of studies have proven the enhanced the CTC capture performance using nanotopographic biomaterials, it remains unclear how the nanotopographic cues could contribute to the enhanced CTC adhesion and capture. It is generally believed that the ECM can accommodate cells through cell adhesion by the mediation of molecules known as integrins [112]. Integrins are heterodimeric transmembrane proteins that have decisive roles in cell-ECM adhesions. Researches have well documented the bidirectional mediation of integrins for cells adhesion in surrounding ECM [113,114]. Initial biding of integrin to adhesive molecules will intrigue intracellular adjustment of adaptor proteins, such as focal adhesion kinase (FAK) that will enhance the affinity between integrins and ECM (Fig. 2). As the binding of integrin to adhesive molecules increase, more adaptor and signaling proteins will be recruited to further enhance the cell-ECM adhesion, known as focal adhesion (FA). FA is susceptible to the nanoscale variation of substrate topography when the head of integrin heterodimer exceeds a threshold (about 20 nm in diameter), because the change of integrin conformation and clustering will lead to the change of adaptor and signaling proteins in FA [69]. Similar to the effect of ECM rigidity, nanotopographic substrates might strongly inference phosphorylation of FAK and in turn regulate FA formation [115]. It is highly possible that FAK was not only responsive to but also required for nanotopography sensitivity and subsequent cellular responses. Therefore, adhesion of CTCs on nanotopographic substrates, which is largely enhanced by forming FA is strongly affected by the surface topography through the conformation and clustering change of integrins. Studies of the effects of nanotopography on the formation of FA were congruent that it was a smaller nanorough feature rather than a greater one that prompted FA formation [116-118].

Furthermore, integrin and FA proteins form a signaling complex that relays signals from integrin to control downstream Rho GTPase activities [119]. RhoA/ROCK signaling will affect the structural quality of filamentous actin cytoskeleton (CSK) and contractile activity of nonmuscle myosin (NMMII) motor proteins to apply mediation on integrin-regulated adhesion signaling [120]. Recently, studies of mechanotransduction have implied the integrating role of actin CSK and its integrity for multitude of upstream signal relayed from extracellular mechanical cues, such as nanotopography [121]. Disorganized F-actin CSK and the resultant changes in CSK contractility observed in cells cultured on nanoroughened further confirmed this idea [122]. Previous studies have suggested that NMMIIA-dependent CSK contractility is a key mediator of the mechanotransduction processes in different types of stem cells [96,122]. Recent results indicated the subcellular organizations of NMMIIA were distinct between cells on the smooth and nanoroughened surfaces [122]. Together, it appeared that nanotopography might regulate cell behaviors through its direct effect on the local molecular arrangement, phosphorylation and activation of FAs that might in turn regulate the spatial organization of NMMIIA-mediated CSK contractility, and thus the cell-ECM adhesion [122].

Integrins are the primary receptors of cells adhering to the ECM, and the constitutive integrin trafficking (activation/clustering, endocytosis, and recycling) play key roles in various cellular processes [113,123]. Although no direct evidence have been provided yet, from various preliminary studies, we speculate that the nanotopography cue significantly influences the integrin activation and endocytosis, and in turn directly regulate interactions with the ECM and proteins involved in adhesion signaling, integrin-linked kinase, FAK phosphorylation, and Rho GTPase activities [124].

Apart from the nanotopographic substrate itself facilitating the cell adhesion, the overall performance of CTC capture using nanotopographic surfaces is also partly depend on the relatively density of capture agent modified on the substrates. Because of the high surface to volume ration of nanotopographic substrates, more dense capture agents can be immobilized and enhance the capture efficiency. And because of the higher density of binding, CTCs are more likely to be stable at the place of being captured rather than being washed away when required higher flow rate. Morphology changes of CTCs when captured on nanotopographic substrates compared to flat substrate were observed because of the formation of pseudopodia, which further confirmed enhanced capture performance of nanotopographic surfaces.

Fabrication Techniques for Nanotopography Preparation.

The enhanced capture of CTCs using nanotopographic substrates and their availability for viable CTC release encourage us to explore potential nanostructures that may exhibit better performance. Before success in applying nanotopographic substrates for the CTC capture and release, it is critical to grasp the general ideas of nanofabrication methods for generating nanotopographic surfaces with different sizes, shapes and dimensions. The development of nanotechnology equip us with various tools and methods for nanotopography fabrication. Overall, techniques used to generate nanotopographic surfaces can be classified into top down—lithography and pattern transfer, and bottom up—surface roughening and material synthesis approaches (Fig. 3 and Table 2).

Lithography is a collection of methods for transferring predefined patterns on masks to substrates using like photolithography and electron beam lithography (EBL). Photolithography is extensively used to prepare micro and submicron scale substrates by using ultraviolet (UV) light exposure to transfer patterns from masks to substrates [125]. However, the relatively low achievable resolution for nanostructure patterning limits its applications for fabrication small structures (i.e., structures of tens of nanometers) [126]. EBL is developed to improve the patterning resolution by applying electron beams and X-rays instead of UV that are capable of creating nanostructures with feature sizes within 10 nm [127]. EBL-fabricated nanotopographic substrates have been used to many cellular studies [128,129]. However, the low processing throughput and high cost might prevent it from large scale sample processing like CTC capture. Different from photolithography and EBL, colloidal lithography is a method using randomly deposited nanoparticles on substrates to act as mask to create pattern for the etching process [130-132]. Simple and less consuming as it is, colloidal lithography is not able to create perfectly controllable patterns. Using colloidal lithography, QWN was fabricated and used for CTC capture [79].

Pattern transfer including nano-imprinting and replica molding is applying nanostructured stamp to transfer existing patters from stamp to substrates. Pattern transfer is cost-effective and suitable for high throughput fabrication. Nano-imprinting is a nanofabrication technique that using thermoplastics to duplicate the patterns in hard stamp by hot embossing [133,134]. Patterns in hard stamps are transferred to thermoplastics by applying pressure and heat between stamps and thermoplastics. This technique can achieve high resolution but have a high requirement for equipment [135,136]. Thermoplastics like poly (methyl methacrylate) and polyurethane acrylate have been used for nano-imprinting [137,138]. Another pattern transfer techniques named replica molding, which realizes the pattern transfer by using elastomeric polymer, like PDMS to replicate patterns from stamp after baking from liquid state to solid state under high temperature to retain the structures in stamp [139,140]. Compared to nano-imprinting, replica molding can be carried out using regular experiment settings.

Different from lithography and pattern transfer which retain accurate predefined patterns, surface roughening through physical or chemical etching process is a technique used to generate random nanotopographic surface, even in a large surface area [122,141,142]. One of the approaches to achieve surface roughness is chemical etching. In chemical etching, etchants will react with substrates in an anisotropic way and remove materials accordingly near the surface to create surface roughness. Silicon wafers and many other biocompatible materials like PLGA and poly-ether-urethane can be etched using chemical etching to generate desired roughness [143]. Another technique extensively applied in semiconductor fabrication to generate nanotopographic surfaces is RIE. Silica-based glass will be activated for etchant after the bombardment from reactive ion species generated by SF6 and C4F8 gases. The roughness of the glass can be controlled by adjusting RIE duration and is compatible with photolithography to generate patterned roughness area [122].

Another straightforward way for generating nanotopography is synthesizing or depositing nanomaterials on the substrates. Electrospinning is extensively employed to produce nanofibrous constructs for a variety of applications [144,145]. Utilizing electrical forces between nozzle and collector with simply experimental settings, electrospinning can generate nanofibers with different diameters from a variety of polymers such as PLGA and polyaramid [146,147]. The spatial arrangement of fibers on substrates can also be arranged. Nanofibers can be advantageous for CTC capture in that fabricated nanofibrous constructions will greatly mimic the structure and spatial arrangement of ECM [148]. CVD is another materials deposition method that materials in a high pressure and high temperature condition will react or decompose on the substrates to form nanostructures. Using CVD, SiNWs were fabricated and employed for further studies [149,150]. Thermally induced phase separation can be used to form polymeric foam that appears as nanostructured scaffolds [151,152]. Phase separation is realized by subliming solvent in which polymers concentrated to remove some chemicals and leaves nanotopography on the substrates. Foams of different morphologies can be generated by controlling polymer concentration and phase separation temperature or using different polymers and solvents.

Conclusions

CTCs are important for the prediction and monitoring of cancer progress and are potential “biopsy” for cancer therapy. However, the capture of CTCs encounter great challenges in the past years not only because that CTCs are extremely rare in bloodstream but also because the heterogeneity of CTCs that the biomarker expression, size, elasticity, and electric properties among each CTC are different. Counting CTC number in patient's blood has been commonly used to identify and predict the cancer progression. However, there is concern that the blood samples obtained from blood vein and the aliquots of sample we use to isolate CTCs may not statistically represent the real physical status. It is possible that sample size and sample probability would generate influence on the accuracy of CTC enumeration and analysis [153]. Besides, despite some improvements have been made, the release of viable CTCs remains difficult. Nanotopographic substrates are promising for the isolation of CTCs with enhanced capture performance and there are many nanotopography preparation methods that enable us to prepare desired nanostructures for CTC detection. Cells prefer to the nanotopographic substrates because of the similarity between nanostructures and ECM. Nevertheless, most of these nanotopography-enhanced approaches did not prove clinical utility. High throughput and purity are still challenges for these devices to be applicable for clinical practice. Although the nanorough glass substrate without functionalization of antibody achieved high throughput and is good for in situ culture of CTCs, further studies on utility of this device on patient samples are desired. In addition, nonspecifically capture of blood cells is still an obstacle for further improving isolation purity. The combination of nanostructured surfaces with other cells separation methods are suggested and may throw light on the capture of CTCs.

Figures

Nanotopography-enhanced capture of CTCs in microfluidic chip. (a) Nanopillar array was applied to be the substrate of microfluidics chip and was functionalized with antibody, (b) label-free nanorough surface was embedded in the microfluidics chip, (c) nanofiber-embedded microfluidics chip functionalized with aptamer, and (d) functionalized graphene oxide nanosheets in microfluidic chip.

Integrins mediate cell adhesion with ECM. (a) Integrin activation triggering a shift from the low-affinity conformation (bent with a closed head) toward the intermediate affinity (extended with closed head) and high affinity (extended with open head) conformation, (b) endocytosis of β1 integrin heterodimers in their active conformation near the front of the cell, and form early endosomes, and (c) integrins are recycled to the membrane and reassemble a new FA.

Nanotopographic substrates preparation methods. (a) EBL is used to fabricate well controlled nanopattern with high resolution, (b) electrospinning is the most often used technique to fabricate nanofibers, (c) RIE are capable of generating nanotopographic substrates with different nanoroughness, and (d) nano-imprinting can be used to achieve high resolution nanofabrication over large surface.

Copyright in the material you requested is held by the American Society of Mechanical Engineers (unless otherwise noted). This email ability is provided as a courtesy, and by using it you agree that you are requesting the material solely for personal, non-commercial use, and that it is subject to the American Society of Mechanical Engineers' Terms of Use. The information provided in order to email this topic will not be used to send unsolicited email, nor will it be furnished to third parties. Please refer to the American Society of Mechanical Engineers' Privacy Policy for further information.

Shibboleth is an access management service that provides single sign-on protected resources.
It replaces the multiple user names and passwords necessary to access subscription-based content with a single user name and password that can be entered once per session.
It operates independently of a user's location or IP address.
If your institution uses Shibboleth authentication, please contact your site administrator to receive your user name and password.